`
`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
`ENZYMOTEC - 1023
`
`
`
`Bull. Fae. Fish. Hokkaido Univ.
`33(4), 398-404. 1987.
`
`Prediction of Relative Retention Value of the Individual
`
`Molecular Species of Diacyl Glycerolipid on
`
`High Performance Liquid Chromatography
`
`Koretaro TAKAHASHI‘ and Tsugihiko HIRANO"
`
`Abstract
`
`The relative retention value of the individual molecular species of aeetyldiglyceride
`derived from phosphetidylcholine on reverse phase high performance liquid chromatography
`was considered to be dependent principally on the addition theorem of chemical potentials of
`the two fatty acid residues.
`It was demonstrated that the chemical potential of each fatty acid residue is equivalent
`to the relative retention potential of each fatty acid residue; and that the addition of the
`relative retention potentials of the two fatty acid residues denoted the logarithm of the
`relative retention value, the relative retention time of each individual molecular species of
`acetyldiglyceride.
`
`Martin formulated the formulae" :
`
`‘A#B/R'T:A#A/R'T+AflX/R'T
`[2]
`10g( 573/011) = Z1ax/ R - T
`where A and B are members of a. homologous series differing by the functional group
`X ; A/1X is the difference in chemical potential of the group X in the chromatogra-
`phic system. R is the gas constant; and T is the absolute temperature.
`If we
`consider am as the partition eoeflicient of the standard molecular species and (13 as
`the partition coeflicient of each molecular species,
`(1,,/gr,‘ will correspond to the
`relative retention value, the relative retention time (RRT ) of each molecular species.
`So from formula [2],
`
`log (aa/aA):log RRT—,{]px/R- T
`should hold. Under most of the chromatographic system, T is constant. Therefore,
`1/R- T will also be constant.
`
`log RRT oc Apx
`From the aforementioned aspect, formula [1] can be rewritten as follows:
`A/1B=A#A ] A1’-ix
`
`[3]
`
`[4]
`
`This formula [4] can be expanded to cover
`[5]
`AIUJC 7 AIIFA. + A#1~‘A,
`where D67 is the diacylglycerolipid and FA, and FA2 are the fatty acid residues of
`DG. From formulae [3], [4] and [5],
`
`Laboratory of Food Chemistry 1, Faculty of risiwm, Hokkmiio University
`(21ti‘§fi7C§"«7.l€E5Y¢‘F'{i§iIxiFzii:7:i'5§'$"§iE)
`Hitachi Holclcai Semicmiducrzrr Ltd.
`(H1kfit5nvfl9fl®mL%)
`
`000002
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`
`
`
`TAKAHASIII & HIRANOZ Prediction of glycerolipid on HPLC
`
`[6]
`log RRT,,G=log .RRTFA,+log RRT,-A,
`So by calculating the relative retention potential of the individ-
`can be obtained.
`ual fatty acid residue, it is possible to predict the relative retention value (;—. RRT)
`of the individual molecular species of DG.
`This idea was demonstrated by comparing the theoretically determined relative
`retention potential of the individual molecular species of DG and also the empiri-
`cally determined one.
`
`Methods
`
`The RRT data of acetyldiglyceridem’, obtained by acetylation subsequent to
`phospholipase C hydrolysis of soybean phosphatidylcholine (PC), egg yolk PC, and
`PC from five kinds of fish muscle were reviewed. The relative ‘retention potential
`of each fatty acid residue of the acetyldiglyceride, i.e., the DG, was calculated as
`follows:
`
`From the identified monoacid DG molecular species,
`log RRTF,,r:—(log RRTDC)/2
`and from the identified diacid DG molecular species,
`
`log RRT,..,h::log RRTDG»~-log RRT”,
`(Unknown)
`(Known)
`
`log RR7'FA,,:log RRTDG—log RRTMI
`(Unknown)
`(Known)
`
`The theoretically determined relative retention potential of the individual DG
`molecular species that denotes log RRTD,;=log RRT”1+log RRT,-AZ (each term is
`calculated from formulae [7],
`[8] and [9]) was compared with the empirically
`determined one.
`
`The “Q” value examination proposed by Dean and Dixon“ was employed in
`order to minimize the error of RRT.
`
`Results
`
`Table 1 shows the theoretically determined relative retention potential of each
`fatty acid residue of the DGS that have been reviewed”’. For example, from the
`RRT data” of (22 : 6)(22 : 6), that is, 44.1, 44.2, 44.3, 44.6, 46.2, all of these could
`be employed for
`the mean value calculation according to the “Q” value
`examination".
`In this case, the mean value of RR T would become 44.6 and the
`relative retention potential of 22 : 6 fatty acid residue was calculated as (log 44.6)/
`220.8247 (See formula
`In the same way, the relative retention potential of
`20 : 5 fatty acid residue was calculated as (log 37.3) /2 =0.7859, therefore, the relative
`retention potential of (20 : 5)(22 : 6) can be predicted as 0.8247 +0/785921.611.
`The actual relative retention potential of (20 : 5)(22 : 6) was 1.613, as shown in Table
`2. The relative error of the retention potential will be (1.613—1.611)/1.611:0.12%
`in case of (20 : 5)(22 : 6). A retention potential of 16:0 was calculated as follows.
`The mean value of the RRT of (16 : 0)(20 : 5) was 92.7“); and, the relative reten—
`tion potential of this molecular species was log 92.721967. Accordingly, from
`—-. 399 ~
`
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`
`
`
`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`Table 1. Relative retention potential of the fatty acid residues
`of acetyldiglyceride on reverse phase high performance
`liquid chromatography.‘
`
`Fatty acid
`residue
`
`14 :
`15 I
`16:
`
`17 :
`18 ;
`20 :
`
`16:
`17:
`13;
`20:
`24:
`
`potential
`1.023
`1.102
`1.181
`
`1.229
`1.347
`1.434
`
`0.9935
`1.081
`1.142
`1.313
`.
`
`resldue
`16 2 2
`17 2 2
`18 2 2
`
`(1)6
`
`(03
`10' : 3
`""20 : 4 we
`:
`
`:
`22 : 5
`22 ; 6
`
`1
`
`:03
`
`0.9757
`
`0.8580
`0.9090
`1.027
`
`0.7959
`0.9261
`0.3247
`
`' Relative retention time of (16:0)(22 : 6) is regarded as 100.
`Equipment, Hitachi 638430; Shodex RI monitor; column,
`LiChros0rb
`RP»—18
`(250><8 mm; tandem); solvent,
`ism
`propan0l/acetone/mcthano1/acetonitrile
`(l :
`l
`: 3 : 4,
`v/V) ;
`flow, 1.5 ml/min; column temperature, ambient.
`
`formula [8], the relative retention potential of 16:0 can be calculated as 1.967-
`0.7859: 1.181.
`Incidentally, the relative retention potential of (16 : 0)(22 : 6) can be
`predicted as 1.181 ~|-0.8247: 2.006 and the actual relative retention potential of (16 :
`0)(22 : 6) was 2.000 (See Table 2). All of the relative retention potentials of the
`fatty acid residues in Table 1 were calculated in the same manner. From the
`calculated retention potential of the fatty acid residues shown in Table‘1,
`the
`relative retention potential of the individual molecular species of DG was theoreti-
`cally predicted and it was compared with the empirically determined one as shown
`in Table 2. Though there are small errors between the theoretically determined
`retention potential and the empirically determined one, the validity of this idea is
`well demonstrated.
`
`Discussion
`
`and [2] should hold
`As it is obvious from the theory of Martin”, formulae
`for all of the partition chromatographic systems.
`It then follows that formulae [6],
`[7], [8] and
`should also hold for all of the partition chromatographic systems of
`DG lipid molecular species regardless of the analytical condition employed for the
`chromatography. By introducing the RR7‘ data of the dinitrobenzoyl derivatives
`from PC presented by Takamura at al.“ and Kito et al.“’, instead of the acetyl
`derivatives that have been discussed in this study, it was also demonstrated that the
`theoretically determined relative retention potential of this derivative coincides well
`with the empirically determined one as shown in Table 3. This shows that not only
`—400—
`
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`000004
`
`
`
`TAKAHASIII & HIRANOZ Prediction of glycerolipid on HPLC
`
`Table 2. Comparison of the predicted and the empirically determined relative retention
`potential of the individual molecular species of ucetyldiglycericle.‘
`
`Molecular species
`of acetyldiglyceride
`
`Predicted relative
`retention potential
`
`Empirically determined
`relative retention
`potential
`
`.
`
`:5)
`(20:
`:6)
`(20 :
`:
`(22:
`'
`:
`(18 :
`:0
`(20:
`1
`(22 :
`:
`(20:
`:
`(22:
`:
`(18:
`:
`(16:
`:
`(18:
`:
`(14:
`(16: l)(22:
`(18 : 2)(18:
`(20 2 4)(22 :
`(14 : o)(22:
`(22 : 4~)(22:
`(22 : 5)(22:
`(18:2)(2():
`(15 : 0)(20;
`(17 : 2)(20:
`(13 : 2)(22:
`(17: 1)(22:
`(16 : 1)(20:
`(17: 2)(22:
`(16 : 1)(22;
`(15 2 O)(22:
`(18 : 1)(20:
`(14 : 0)(22:
`(18 2 2)(18:
`(18 : 1)(22 :
`(16 : 0)(20:
`(16: 1)(1s:
`(18:1)(18:.
`(14 : 0)(18:
`(16 2 0)(22:
`(16 2 O)(18:
`(16 : 0)(16:
`(18:1)(20:
`(17 : 0)(22:
`
`1.572
`1.611
`1.649
`1.683
`1.695
`1.712
`1.734
`1.751
`1.762
`1.784
`1.800
`1.309
`1.823
`1.834
`1.835
`1.848
`1.852
`1.852
`1.885
`1.888
`1.894
`1.906
`1.906
`1.908
`1.911
`1.925
`1.927
`1.928
`1.949
`1.951
`1.966
`1.967
`1.974
`1.999
`1.999
`2.006
`2.039
`2.043
`2.051
`2.053
`
`1.572
`1.613
`1.649
`1.688
`1.719
`1.712
`1.722
`1.746
`1.788
`1.784
`1.825
`1.309
`1.824
`1.848
`1.869
`1.843
`1.852
`1.394
`1.917
`1.391
`1.894
`1.900
`1.906
`1.918
`1.895
`1.900
`1.927
`1.945
`1.915
`1.981
`1.966
`1.967
`2.000
`1.999
`2.000
`2.000
`2.030
`2.043
`2.064
`2.067
`
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`
`
`
`Bull. Fae. Fish. Hokkaido Univ. 38(4), 1987.
`
`Table 2
`
`(continued).
`
`Molecular species
`of wcetyldiglyccridc
`
`Predicted relative
`retention potential
`
`Empirically determined
`relative retention
`potential
`
`Relative
`error {"/D)
`
`(18 : 1)(22 : 5)
`(16 : 0)(20 : 4)
`(20 ; 1)(20 : 5)
`(16 ; 0)(22 ; 5)
`(18:
`: 2)
`(13:
`: 5)
`(20 ;
`:6)
`(1.8:
`: 1)
`(16:
`:2)
`(14 :
`: 1)
`(l8:0)(22:6)
`(16 : 0)(16: 1)
`(13 ; 0)(l8 ; 3)
`(17 :0)(1a : 2)
`(16 :0)(22 : 4)
`(15 : 0)(18: 1)
`(13 : 0)(20: 4)
`(16 : 0)(17: 1)
`(18 ; 1)(13: 1)
`1
`:2)(20 : 1)
`:o)(1s: 1)
`:0)(20; 1)
`:0)(16 : 0)
`:0)(22 : 4)
`:1)(20 : 5)
`:2)(20 : 0)
`:0)(1s : 1)
`: 0)(20 : 1)
`
`-
`
`‘
`
`2.068
`2.090
`2.099
`2.107
`2.117
`2.133
`2.133
`2.140
`2.157
`2.165
`2.172
`2.179
`2.205
`2.204
`2.203
`2.244
`2.256
`2.262
`2.283
`2.289
`2.323
`2.336
`2.362
`2.374
`2.424
`2.460
`2.489
`2.494
`
`2.067
`2.090
`2.093
`2.080
`2.137
`2.137
`2.138
`2.137
`2.148
`2.155
`2.172
`2.172
`2.134
`2.204
`2.126
`2.245
`2.254
`2.245
`2.281
`2.274
`2.321
`2.335
`2.328
`2.283
`2.424
`2.460
`2.480
`2.480
`
`0.05
`*4:
`0.05
`1.28
`0.94
`0.19
`an
`0.14
`0.42
`0.46
`*4
`0.32
`0.95
`*9:
`3.71
`0.04
`0.09
`0.75
`0.09
`0.66
`0.09
`:0
`1.44
`3.83
`**
`*1:
`
`‘ Relative retention time of (16 : [))(22 :6) is regarded as 100.
`Analytical conditions are the same as in Table 1.
`" Relative retention potentials of the fatty acid residues shown in Table 1 are calculated
`from the log R.RTs of these molecular species as explained in the text.
`
`chromatographic conditions such as the column, the solvents, the analytical tempera-
`ture (as long as it is constant), but also the type of derivative does not matter for the
`proposed idea, while the value itself of the relative retention potential of each fatty
`acid residue has to be calculated each time for each chromatographic condition.
`The idea proposed in thm study can be expanded to cover the paflfifion
`chromatographic system of triglyceride. This will be reported at a later date.
`
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`
`
`
`TAKAHASHI & IIIIRANO : Prediction of glycerolipid on HPLC
`
`Table 3. Comparison of the predicted and the empirically determined relative retention potential
`of the individual molecular species of dinitrobenzoyl derivative of diacylglycerolipid i.e.
`diglyceride (D ).'
`
`Molecular species of
`dinitrobcnzoyl
`derivative of D0-
`
`Predicted relative
`retention potential
`
`lmpiricully determind
`relative retention
`potential
`
`2.030
`2.225
`2.233
`2.243
`2.414
`2.431
`2.529
`2.547
`2.564
`
`2.190
`2.210
`2.241
`2.260
`2.305
`2.305
`2.356
`2.534
`
`2.090
`2.225
`2.233
`2.243
`2.405
`2.431
`2.523
`2.555
`2.533
`
`2.190
`2.223
`2.230
`2.260
`2.305
`2.305
`2.356
`2.534
`
`2.243
`2.332
`2.394
`2.400
`2.544
`2.564
`2.571
`2.583
`. 1...
`
`2.243
`2.332
`2.339
`2.490
`2.544
`2.566
`2.571
`2.533
`jj._____._._ A
`
`ii (16: 1)(22 ; 6)
`(13: 1)(22 : 6)
`(16 : 1)(16: 1)
`(16 : 0)(22 ; 6)
`(13: 1)(22 : 4)
`(16 : 0)(22 : 4)
`(13 : 1)(1s: 1)
`(16 : 0)(13: 1)
`(16 : o)(16 : 0)
`
`(.16: 1)(20 : 5)
`(13 : 2)(20 ; 5)
`(16: 1)(22 ; 6)
`(13 : 2)(22 : 6)
`(10 : 0)(20 : 5)
`(13: 1)(20: 5)
`(16 : o)(22 : 6)
`(16 : O)(16:
`
`:2)(18 ;
`16: 0)(20 :
`18: 1)(1s :
`18 : 0)(2o;
`13: 1)(1s :
`6: 0)(13 ;
`:0)(13:
`16 : O)(16:
`
`1 ( <
`
`.( ( 4 1
`
`._...:L__
`
`‘ Relative retention time of (12 :0)(12 : 0) is regarded as 100.
`" Relative retention potentials of the fatty acid residues are calculated from the log RRT3 of
`these molecular species.
`A: Calculated from the data. of Takamuro at 11.1.5’
`Equipment, Hitachi 655-15 ; Ilitnchi 638 41 UV monitor; column, Ultrasphere ODS (250x
`4.6 mm) ; solvent, aeetonitrile/isopropanol (4: 1, v/v) ; flow, 1.0 ml/min; column tempera:
`ture, 25'C.
`: Same condition as A except that the solvent system used is methanol/isopropunol (95 : 5,
`v/v).
`: Calculated from the data. of Kim et al.”
`Same condition as A though the analyzed date is diflerent.
`
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`
`
`
`Bull. Fac. Fish. Hokkaido Univ. 38(4), 1987.
`
`Acknowledgment
`
`Thanks are due to Professors M. Hatano, T. Takagi and K. Takama. for their
`advices.
`
`References
`
`Some theoretical aspects of partition chromatography. Biochem. Soc.
`Martin, A.J.P. (1950).
`Sympom, (Cambridge, England). 3, 420.
`(1982). Molecular species of fish muscle
`Takahashi, K., Hirano, T., Takama, K. and Zama, K.
`lecithin. Bull. Japan. Soc. Sci. Fv,'sh.., 48, 1803-1814.
`Takahashi, K.
`(1985). A novel approach for the identification of lipid molecular species.
`Application of high performance liquid chromatography on fish muscle lecithin molecular
`species analysis. Mem. Fac. Fish. Hokkaido Um'v., 32, 245—330.
`Dean, R.B. and Dixon, W.J.
`(1951). Simplified statistics for small numbers of observations.
`Anal. C'hem., 23, 636--638.
`Takamura, H., Narita, H., Urade, R. and Kito, M. (1986). Quantitative analysis of polyenoic
`phospholipid molecular species by high performance liquid chromatography. Lipids, 21,
`356-361.
`Kito, M., Takamura, H., Narita, II. and Urade, R. (1985). A sensitive method for quantita-
`tive analysis of phospholipid molecular species by high performance liquid chromatography.
`J. I§'iochem., 98, 327»—-33.1.
`
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